Among the major food allergies, peanut, egg, and milk are the most common. The immunochemical detection of food allergens depends on various factors, such as the food matrix and processing method, which can affect allergen conformation and extractability. This study aimed to (1) develop matrix-specific incurred reference materials for allergen testing, (2) determine whether multiple allergens in the same model food can be simultaneously detected, and (3) establish the effect of processing on reference material stability and allergen detection. Defatted peanut flour, whole egg powder, and spray-dried milk were added to cookie dough at seven incurred levels before baking. Allergens were measured using five commercial enzyme-linked immunosorbent assay (ELISA) kits. All kits showed decreased recovery of all allergens after baking. Analytical coefficients of variation for most kits increased with baking time, but decreased with incurred allergen level. Thus, food processing negatively affects the recovery and variability of peanut, egg, and milk detection in a sugar cookie matrix when using immunochemical methods.
In March 2013 a large shipment of maize, intended for feed was subject of an alert in the Rapid Alert System for Food and Feed of the European Commission (EC) because the aflatoxin B1 (AFB1) level in the load exceeded the EC regulated maximum level of 20 μg/kg. Since the shipment had passed import controls and was already distributed (mainly to German farms), a massive recall followed. The aim of the current study was to investigate questions, raised by authorities and industry, related to the effectivity of EU sampling procedures, the influence of sample homogenisation procedures and sample storage conditions on the test results, and fungal identification as unexpected mycotoxins were identified during this study. The Netherlands Food and Consumer Product Safety Authority seized a shipload of maize in July 2013, suspected to be contaminated with AFB1. The shipload was sampled according to the 2009 and 2013 EC Sampling Regulations to compare the outcomes of both sampling protocols. Mycotoxin analysis of the incremental samples showed high mean levels of AFB1, aflatoxin G1 (AFG1), and ochratoxin A (OTA). Also an extreme inhomogeneous distribution of aflatoxins and OTA was proven. Analysis of samples homogenised according to the slurry method showed improved performance as compared to samples homogenised through dry homogenisation. Sampling and sample homogenisation according to the Regulation from 2013 showed a closer estimate of the ‘true’ AFB1 content as compared to sampling according to the Regulation from 2009. No influence of laboratory storage conditions on AFB1 concentration could be determined. Fungal identification revealed Aspergillus flavus as the main source of AFB1 in this shipment. Infrequent occurrence of Aspergillus parasiticus might have been the source of AFG1. The occurrence of sometimes large amounts of OTA could not be explained, however it was suggested that Aspergillus welwitschiae might have played a role.
Peanut proteins can cause allergenic reactions that can result in respiratory and circulatory effects in the body sometimes leading to shock and death. The determination of peanut proteins in foods by analytical methods can reduce the risk of serious reactions in the highly sensitized individual by allowing for the detection of these proteins in a food at various stages of the manufacturing process. The method performance of 4 commercially available enzyme-linked immunosorbent assay (ELISA) kits was evaluated for the detection of peanut proteins in milk chocolate, ice cream, cookies, and breakfast cereals: ELISA-TEK Peanut Protein Assay, now known as “Bio-Kit” for peanut proteins, from ELISA Technologies Inc.; Veratox for Peanut Allergens from Neogen Corp.; RIDASCREEN Peanut Kit from R-Biopharm GmbH; and ProLisa from Canadian Food Technology Ltd. The 4 test kits were evaluated for accuracy (recovery) and precision using known concentrations of peanut or peanut proteins in the 4 food matrixes. Two different techniques, incurred and spiked, were used to prepare samples with 4 known concentrations of peanut protein. Defatted peanut flour was added in the incurred samples, and water-soluble peanut proteins were added in the spiked samples. The incurred levels were 0.0, 10, 20, and 100 μg whole peanut per g peanut protein per g food; the spiked levels were 0.0, 5, 10, and 20 μg peanut protein per g food. Performance varied by test kit, protein concentration, and food matrix. The Veratox kit had the best accuracy or lowest percent difference between measured and incurred levels of 15.7% when averaged across all incurred levels and food matrixes. Recoveries associated with the Veratox kit varied from 93 to 115% for all food matrixes except cookies. Recoveries for all kits were about 50% for cookies. The analytical precision, as measured by the variance, increased with an increase in protein concentration. However, the coefficient of variation (CV) was stable across the 4 incurred protein levels and was 7.0% when averaged across the 4 food matrixes and analytical kits. The R-Biopharm test kit had the best precision or a CV of 4.2% when averaged across all incurred levels and food matrixes. Because measured protein values varied by test kit and food matrix, a method was developed to normalize or transform measured protein concentrations to an adjusted protein value that was equal to the known protein concentration. The normalization method adjusts measured protein values to equal the true protein value regardless of the type test kit or type food matrix.
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